Anoikis-apoptotic cell death triggered by loss of extracellular matrix (ECM) contacts-is dysregulated in many diseases. Anoikis resistance contributes to the development and progression of cancer, thus marking aggressive tumorigenic transitions. In oral/head and neck squamous cell carcinoma (HNSCC), anoikis resistance induces more aggressive tumors. Given that oral SCC, the most common malignant oral neoplasm, accounts for 90% of all oral malignancies and has a poor 5-year survival rate that has not changed in decades (http://seer/cancer.gov), this underscores the need to identify novel therapeutic targets for HNSCC. Identifying markers of aggressive tumorigenesis and thus anoikis resistance could yield potentially novel therapeutic targets for HNSCC. Mass spectrometry-based metabolomics offers an innovative non-invasive platform for the development of marker panels that are characteristic of disease phenotypes or cellular processes that are readily measured in biofluids/tissues. We recently performed NMR based metabolomic analysis of primary and metastatic HNSCC human specimens and found elevated levels of several metabolites (Somashekar et al., 2011). These metabolites were associated with highly active glycolysis and glutaminolysis, increased amino acid influx into the Krebs cycle, and altered energy metabolism, membrane choline phospholipid metabolism, and oxidative/osmotic defense mechanisms. Using NMR spectroscopy, several groups also generated a list of similar metabolites differentially expressed across tissue and blood samples from head and neck cancer patients, (Mukheriji et al., 1997; Tiziani et al., 2009; El-Sayed et al., 2002). In complementary preliminary data where we now used mass spectrometry analyses of primary HNSCC, we identified several metabolites that were differentially and significantly elevated in HNSCC compared to normal tissues. Importantly, these metabolites were also elevated in metastatic HNSCC and in mouse tumors derived from anoikis-resistant HNSCC cells. These metabolites included kynurenine, serine, glutathione, and glutamate/glutamic acid. Since these metabolites have also been implicated in cancer metabolic pathways they constitute potential candidates for a marker panel of aggressive HNSCC. Furthermore, compared to normal oral keratinocytes, HNSCC cells also show elevated levels of glutamic acid. Elevated glutamic acid levels were suppressed in HNSCC cells by chemically inhibiting the enzyme that catalyzes the formation of glutamic acid levels, glutaminase. Inhibiting glutamic acid levels in these cells led to suppression of anoikis resistance/an aggressive phenotype in vitro. In addition, significantly elevated expression levels of glutaminase were highest in the metastatic tissues compared to primary HNSCC and normal tissues. These findings suggest that these metabolites, including glutamic acid may be markers for the transition to a more aggressive phenotype in cancer, including the acquisition of anoikis resistance. Our data support the concept that identification of a marker panel of metabolites present in aggressive HNSCC, a phenotype that emanates in part from anoikis resistance, may be useful as a new diagnostic tool and for identification of novel therapeutic targets for aggressive HNSCC. Thus, we hypothesize that aggressive states in HNSCC and anoikis-resistant HNSCC cells/tissues exhibit a metabolomic profile defined by increased levels of key metabolites that may include kynurenine, glutamic acid, serine, and glutathione. These metabolites mark aggressive tumorigenesis, the transition to anoikis resistance, and may promote anoikis resistance and an aggressive phenotype in vivo. These studies will help establish a signature metabolome or a panel of key metabolites for aggressive HNSCC and anoikis resistance plus validate the functional contribution of these metabolites in this process.